U.S. patent application number 10/867420 was filed with the patent office on 2004-12-02 for prewetting stop flow test strip.
This patent application is currently assigned to PRAXSYS BIOSYSTEMS, LLC.. Invention is credited to DiNello, Robert K., Polito, Alan J., Quan, Stella S..
Application Number | 20040241882 10/867420 |
Document ID | / |
Family ID | 25239956 |
Filed Date | 2004-12-02 |
United States Patent
Application |
20040241882 |
Kind Code |
A1 |
DiNello, Robert K. ; et
al. |
December 2, 2004 |
Prewetting stop flow test strip
Abstract
A test strip and method for detecting an analyte present in a
sample. The test strip comprising: a buffer addition zone to which
a buffer may be added; an absorbent zone proximal to the buffer
addition zone; one or more test zones distal to the buffer addition
zone, at least one of the test zones including a first analyte
binding agent immobilized therein which is capable of binding to
the analyte to be detected; a terminal buffer flow zone distal to
the one or more test zones, the absorbent zone being positioned
relative to the buffer addition zone and having an absorption
capacity relative to the other zones of the test strip such that
when a volume of buffer within a predetermined buffer volume range
for the test strip is added to the buffer addition zone, a distal
diffusion front of the buffer diffuses from the buffer addition
zone to a distal diffusion point within the terminal buffer flow
zone and then diffuses proximal relative to the one or more test
zones; and a sample addition zone distal to the terminal buffer
flow zone to which a sample may be added.
Inventors: |
DiNello, Robert K.;
(Hayward, CA) ; Polito, Alan J.; (Danville,
CA) ; Quan, Stella S.; (Moraga, CA) |
Correspondence
Address: |
ROBINS & PASTERNAK
1731 EMBARCADERO ROAD
SUITE 230
PALO ALTO
CA
94303
US
|
Assignee: |
PRAXSYS BIOSYSTEMS, LLC.
|
Family ID: |
25239956 |
Appl. No.: |
10/867420 |
Filed: |
June 14, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10867420 |
Jun 14, 2004 |
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09823868 |
Mar 30, 2001 |
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6767710 |
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Current U.S.
Class: |
436/518 |
Current CPC
Class: |
B01L 2300/0867 20130101;
Y10S 436/81 20130101; Y10S 435/81 20130101; Y10S 436/817 20130101;
Y10S 435/974 20130101; Y10T 156/1089 20150115; Y10S 435/97
20130101; B01L 3/5023 20130101; Y10S 436/811 20130101; Y10S 436/808
20130101; B01L 3/5055 20130101; Y10S 435/975 20130101; G01N 33/558
20130101; Y10S 436/807 20130101; G01N 33/54386 20130101; Y10S
436/805 20130101 |
Class at
Publication: |
436/518 |
International
Class: |
G01N 033/543 |
Claims
What is claimed is:
1. A test strip adapted to receive a sample and detect an analyte
therein, the test strip comprising: a buffer addition zone to which
a buffer may be added; an absorbent zone proximal to the buffer
addition zone; one or more test zones distal to the buffer addition
zone, at least one of the test zones including a first analyte
binding agent immobilized therein which is capable of binding to
the analyte to be detected; a terminal buffer flow zone distal to
the one or more test zones, the absorbent zone being positioned
relative to the buffer addition zone and having an absorption
capacity relative to the other zones of the test strip such that
when a volume of buffer within a predetermined buffer volume range
for the test strip is added to the buffer addition zone, a distal
diffusion front of the buffer diffuses from the buffer addition
zone to a distal diffusion point within the terminal buffer flow
zone and then diff-uses proximal relative to the one or more test
zones; and a sample addition zone distal to the terminal buffer
flow zone to which a sample may be added.
2. A test strip according to claim 1 wherein the test strip further
includes a zone distal to the terminal buffer flow zone which
includes a second analyte binding agent which is capable of binding
to the analyte and diffusing to the one or more test zones.
3. A test strip according to claim 1 wherein the second analyte
binding agent is capable of binding to components in the sample
other than the analyte.
4. A test strip according to claim 1 wherein the second analyte
binding agent does not bind to components in the sample other than
the analyte.
5. A test strip according to claim 1 wherein the second analyte
binding agent is labeled with a detectable marker.
6. A test strip according to claim 1 wherein the second analyte
binding agent is attached to a particle which is capable of
diffusing to the one or more test zones.
7. A test strip according to claim 1 wherein the zone containing
the second analyte binding agent is proximal to the sample addition
zone.
8. A test strip according to claim 1 wherein the zone containing
the second analyte binding agent is the sample addition zone.
9. A test strip according to claim 1 wherein the test strip further
includes a zone distal to the terminal buffer flow zone which
includes a competitive agent which is capable of competing with the
analyte to bind to the first analyte binding agent.
10. A test strip according to claim 1 wherein the competitive agent
binds to the first analyte binding agent and does not bind to
components in the sample other than the first analyte binding
agent.
11. A test strip according to claim 1 wherein the competitive agent
is labeled with a detectable marker.
12. A test strip according to claim 1 wherein the competitive agent
is attached to a particle which is capable of diffusing to the one
or more test zones.
13. A test strip according to claim 1 wherein the zone containing
the competitive agent is proximal to the sample addition zone.
14. A test strip according to claim 1 wherein the zone containing
the competitive agent is the sample addition zone.
15. A test strip according to claim 1 wherein the sample addition
zone is positioned relative to the test zones such that sample
added to the sample addition zone at the same time as buffer is
added to the buffer addition zone reaches the distal diffusion
point after the distal diffusion front of the buffer has diffused
to the distal diffusion point and begun diffusing in a proximal
direction.
16. A test strip according to claim 1 wherein the sample addition
zone is positioned relative to the test zones such that sample
added to the sample addition zone at the same time as buffer is
added to the buffer addition zone reaches the test zones after the
distal diffusion front diffuses proximal relative to the test
zones.
17. A test strip according to claim 1 wherein the predetermined
buffer volume range is between about 10 and 250 L.
18. A test strip according to claim 1 wherein the predetermined
buffer volume range is between about 20 and 200 L.
19. A test strip according to claim 1 wherein the predetermined
buffer volume range is between about 20 and 100 L.
20. A test strip according to claim 1 wherein the predetermined
buffer volume range is between about 40 and 60 L.
21. A test strip according to claim 1 wherein the terminal buffer
flow zone has a length from a proximal end to a distal end of
between about 3 and 10 mm.
22. A test strip according to claim 1 wherein the first analyte
binding agent does not bind to components in the sample other than
the analyte.
23. A test strip according to claim 1 wherein the first analyte
binding agent is selected from the group consisting of antibodies,
engineered proteins, peptides, haptens, lysates containing
heterogeneous mixtures of antigens having analyte binding sites,
ligands and receptors.
24. A test strip according to claim 1 wherein the test zones
further include at least a first control zone with a control
binding agent immobilized therein.
25. A test strip according to claim 1 wherein the test zones
further include a first control zone with a control binding agent
immobilized therein, and a second control zone with a same control
binding agent immobilized therein as the first control zone, the
first and second control zones containing a different amount of the
control binding agent immobilized therein.
26. A test strip according to claim 1 wherein the test zones
further include a first control zone with a control binding agent
immobilized therein, and a second control zone with a same control
binding agent immobilized therein as the first control zone, the
first and second control zones containing about the same amount of
the control binding agent immobilized therein.
27. A test strip according to claim 1 wherein the test zones
further include first and second control zones each with a control
binding agent immobilized therein, the first test zone being
proximal to the test zone including the first analyte binding
agent, the second control zone being distal to the test zone
including the first analyte binding agent.
28. A method for detecting an analyte in a sample comprising:
delivering a buffer to a test strip which causes a distal diffusion
front of the buffer to (a) diffuse in a distal direction to one or
more test zones, at least one of the test zones including a first
analyte binding agent immobilized therein which binds to analyte in
the sample, (b) diffuse to a terminal buffer flow zone distal to
the one or more test zones, change direction and (c) diffuse to a
position proximal to the one or more test zones; delivering a
sample to the test strip at a position distal to the terminal
buffer flow zone, delivery of the sample causing analyte in the
sample to diffuse proximally past the terminal buffer flow zone to
the one or more test zones after the distal diffusion front of the
buffer diffuses proximal to the one or more test zones, the analyte
binding to the first analyte binding agent and becoming immobilized
in the test zones; and detecting the analyte immobilized in the
test zones.
29. A method according to claim 28 wherein the test strip further
includes a zone distal to the terminal buffer flow zone which
includes a second analyte binding agent which is capable of binding
to the analyte, addition of the sample causing the second analyte
binding agent to bind to analyte in the sample, binding of the
analyte to the first analyte binding agent causing the second
analyte binding agent to be immobilized in the test zones, and
detecting the analyte immobilized in the test zones comprising
detecting the second analyte binding agent.
30. A method according to claim 29 wherein the second analyte
binding agent is labeled with a detectable marker and detecting the
second analyte binding agent comprises detecting the detectable
marker.
31. A method according to claim 29 wherein the second analyte
binding agent is attached to a particle and detecting the second
analyte binding agent comprises detecting the particle.
32. A method according to claim 29 wherein the zone containing the
second analyte binding agent is proximal to the sample addition
zone.
33. A method according to claim 29 wherein the zone containing the
second analyte binding agent is the sample addition zone.
34. A method according to claim 28 wherein the sample addition zone
is positioned relative to the test zones such that sample added to
the sample addition zone at the same time as buffer is added to the
buffer addition zone reaches the distal diffusion point after the
distal diffusion front of the buffer has diffused to the distal
diffusion point and begun diffusing in a proximal direction.
35. A method according to claim 28 wherein the sample addition zone
is positioned relative to the test zones such that sample added to
the sample addition zone at the same time as buffer is added to the
buffer addition zone reaches the test zones after the distal
diffusion front diffuses proximal relative to the test zones.
36. A method according to claim 28 wherein the buffer delivered to
the buffer addition zone has a volume between about 10 and 250
L.
37. A method according to claim 28 wherein the buffer delivered to
the buffer addition zone has a volume between about 20 and 200
L.
38. A method according to claim 28 wherein the buffer delivered to
the buffer addition zone has a volume between about 20 and 100
L.
39. A method according to claim 28 wherein the buffer delivered to
the buffer addition zone has a volume between about 40 and 60
L.
40. A method according to claim 28 wherein the buffer delivered to
the buffer addition zone comprises the sample delivered to the
sample addition zone.
41. A method according to claim 28 wherein the buffer delivered to
the buffer addition zone is the same as the sample delivered to the
sample addition zone.
42. A method according to claim 28 wherein the buffer delivered to
the buffer addition zone has substantially the same fluid flow
characteristics within the test strip as the sample delivered to
the sample addition zone.
43. A method according to claim 28 wherein the test zones further
include a first control zone with a control binding agent
immobilized therein, and a second control zone with a same control
binding agent immobilized therein as the first control zone, the
first and second control zones containing a different amount of the
control binding agent immobilized therein.
44. A method according to claim 28 wherein the test zones further
include a first control zone with a control binding agent
immobilized therein, and a second control zone with a same control
binding agent immobilized therein as the first control zone, the
first and second control zones containing about the same amount of
the control binding agent immobilized therein.
45. A method according to claim 28 wherein the test zones further
include first and second control zones each with a control binding
agent immobilized therein, the first test zone being proximal to
the test zone including the first analyte binding agent, the second
control zone being distal to the test zone including the first
analyte binding agent.
46. A method for detecting an analyte in a sample comprising:
delivering a buffer to a test strip which causes a distal diffusion
front of the buffer to (a) diffuse in a distal direction to one or
more test zones, at least one of the test zones including a first
analyte binding agent immobilized therein which binds to analyte in
the sample, (b) diffuse to a terminal buffer flow zone distal to
the one or more test zones, change direction and (c) diffuse to a
position proximal to the one or more test zones; delivering a
sample to the test strip at a position distal to the terminal
buffer flow zone, delivery of the sample causing analyte in the
sample to diffuse proximally past the terminal buffer flow zone to
the one or more test zones after the distal diffusion front of the
buffer diffuses proximal to the one or more test zones, the analyte
binding to the first analyte binding agent and becoming immobilized
in the test zones; and delivery of the sample to the test strip
also causing a competitive agent to diffuse with the sample to the
test zone, the competitive agent being capable of binding to the
first analyte binding agent and thus competing with the analyte for
binding to the first analyte binding agent. detecting the
competitive agent immobilized in the test zones.
47. A method according to claim 46 wherein the competitive agent is
comprised on the test strip in a zone distal to the terminal buffer
flow zone.
48. A method according to claim 47 wherein the competitive agent is
labeled with a detectable marker.
49. A method according to claim 47 wherein the competitive agent is
attached to a particle which is capable of diffusing to the one or
more test zones.
50. A method according to claim 46 wherein the zone containing the
competitive agent is proximal to the sample addition zone.
51. A method according to claim 46 wherein the zone containing the
competitive agent is the sample addition zone.
52. A method according to claim 46 wherein the sample addition zone
is positioned relative to the test zones such that sample added to
the sample addition zone at the same time as buffer is added to the
buffer addition zone reaches the distal diffusion point after the
distal diffusion front of the buffer has diffused to the distal
diffusion point and begun diffusing in a proximal direction.
53. A method according to claim 46 wherein the sample addition zone
is positioned relative to the test zones such that sample added to
the sample addition zone at the same time as buffer is added to the
buffer addition zone reaches the test zones after the distal
diffusion front diffuses proximal relative to the test zones.
54. A method according to claim 46 wherein the buffer delivered to
the buffer addition zone has a volume between about 10 and 250
L.
55. A method according to claim 46 wherein the buffer delivered to
the buffer addition zone has a volume between about 20 and 200
L.
56. A method according to claim 46 wherein the buffer delivered to
the buffer addition zone has a volume between about 20 and 100
L.
57. A method according to claim 46 wherein the buffer delivered to
the buffer addition zone has a volume between about 40 and 60
L.
58. A method according to claim 46 wherein the buffer delivered to
the buffer addition zone comprises the sample delivered to the
sample addition zone.
59. A method according to claim 46 wherein the buffer delivered to
the buffer addition zone is the same as the sample delivered to the
sample addition zone.
60. A method according to claim 46 wherein the buffer delivered to
the buffer addition zone has substantially the same fluid flow
characteristics within the test strip as the sample delivered to
the sample addition zone.
61. A method according to claim 46 wherein the test zones further
include a first control zone with a control binding agent
immobilized therein, and a second control zone with a same control
binding agent immobilized therein as the first control zone, the
first and second control zones containing a different amount of the
control binding agent immobilized therein.
62. A method according to claim 46 wherein the test zones further
include a first control zone with a control binding agent
immobilized therein, and a second control zone with a same control
binding agent immobilized therein as the first control zone, the
first and second control zones containing about the same amount of
the control binding agent immobilized therein.
63. A method according to claim 46 wherein the test zones further
include first and second control zones each with a control binding
agent immobilized therein, the first test zone being proximal to
the test zone including the first analyte binding agent, the second
control zone being distal to the test zone including the first
analyte binding agent.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to lateral flow test strips
and methods of operation for the lateral flow test strips.
[0003] 2. Description of Related Art
[0004] Quantitative analysis of cells and analytes in fluid
samples, particularly bodily fluid samples, often provides critical
diagnostic and treatment information for physicians and patients.
For example, immunological testing methods which take advantage of
the high specificity of antigen-antibody reactions, provide one
approach to measurement of analytes. Kennedy, D. M. and S. J.
Challacombe, eds., ELISA and Other Solid Phase Immunoassays:
Theoretical and Practical Aspects, John Wiley and Sons, Chichester
(1988). This document and all others cited to herein, are
incorporated by reference as if reproduced fully below. Such assays
may also find use in various other applications, such as
veterinary, food testing, or agricultural applications.
[0005] Immunoassays that provide a quantitative measurement of the
amount of an analyte in a sample have previously used complex,
multi-step procedures and expensive analyzers available only in a
laboratory setting.
[0006] Immunochromatographic assays, such as those described in GB
2,204,398A; U.S. Pat. Nos. 5,096,837, 5,238,652, and 5,266,497;
Birnbaum, S. et al., Analytical Biochem. 206:168-171 (1992);
Roberts, M. A. and R. A. Durst, Analytical Chem. 67:482-491 (1995);
and Klimov, A. D. et al., Clinical Chem. 41:1360 (1995), are
simpler, yet do not provide a quantitative measurement of an
analyte. Instead, these immunochromatographic assays detect the
presence (or absence) of an analyte above a defined cutoff level
for the test performed. The lack of a quantitative measurement
limits the usefulness of these assays.
[0007] A variety of disposable diagnostic assay devices have also
been developed. Examples of such devices include, but are not
limited to Cathey, et al, U.S. Pat. No. 5,660,993; International
Publication Number WO 92/12428; Eisinger, et al, U.S. Pat. No.
4,943,522; ;Campbell, et al, U.S. Pat. No. 4,703,017; Campbell, et
al, U.S. Pat. No. 4,743,560; and Brooks, U.S. Pat. No. 5,753,517.
Nevertheless, a need still exists for improved disposable
diagnostic assay devices and methods.
SUMMARY OF THE INVENTION
[0008] Test strips are provided which are adapted to receive a
buffer that prewets the test strip and receive a sample which flows
within the prewet test strip. The test strips are employed to
detect one or more analytes that may be present in a sample.
[0009] According to one embodiment, the test strip comprises a
buffer addition zone to which a buffer is added to prewet the test
strip; an absorbent zone proximal to the buffer addition zone; one
or more test zones distal to the buffer addition zone, at least one
of the test zones including a first analyte binding agent
immobilized therein which is capable of binding to the analyte to
be detected; and a terminal buffer flow zone distal to the one or
more test zones, the absorbent zone being positioned relative to
the buffer addition zone and having an absorption capacity relative
to the other zones of the test strip such that when a volume of
buffer within a predetermined buffer volume range for the test
strip is added to the buffer addition zone, a distal diffusion
front of the buffer diffuses from the buffer addition zone to a
distal diffusion point within the terminal buffer flow zone and
then diffuses proximal relative to the one or more test zones. The
test strip further comprises a sample addition zone that is distal
to the terminal buffer flow zone. When a sample is added to the
sample addition zone, the sample diffuses within the test strip in
a proximal direction across the terminal buffer flow zone, across
the one or more test zones, and ultimately to the absorbent zone.
When the sample traverses the test zones, analyte in the sample is
immobilized in whichever test zone(s) include(s) the first analyte
binding agent bound therein.
[0010] The above described test strip may be used to detect an
analyte in a sample by a direct detection assay or may be used to
detect an analyte in a sample by a competitive assay. When the
assay is a direct detection assay, the amount of analyte in the
sample is measured based on the amount of analyte which is
immobilized in a test zone by a first analyte binding agent bound
therein. When the assay is a competitive assay, the test strip
further comprises a competitive agent which is capable of competing
with the analyte for binding to the first analyte binding agent. In
this instance, the amount of analyte in the sample is measured
based on how much less competitive agent is immobilized in the test
zone by the first analyte binding agent as compared to when a
control is employed as the sample which contains no analyte.
[0011] Control over the above described flow of the buffer within
the test strip (i.e., such that the buffer reaches the terminal
buffer flow zone and reverses the direction of buffer flow within
the terminal buffer flow zone back toward the buffer addition zone
and the absorbent zone) is achieved by controlling the amount of
buffer added to the test strip within a predetermined range
designed to be used with that test strip.
[0012] By adding the sample to the sample addition zone such that
the sample reaches the terminal buffer flow zone after the buffer
has reached the terminal buffer flow zone and has already reversed
direction and is diffusing back toward the absorbent zone, the
sample is able to flow within a prewet test strip, thereby yielding
more accurate and precise results.
[0013] As will be described in greater detail herein, depending on
the layout of the test strip, the buffer may be added before, at
the same time, or after the sample is added to the test strip. For
example, the sample addition zone may be positioned relative to the
test zones such that sample is added to the sample addition zone at
the same time that buffer is added to the buffer addition zone. The
sample addition zone may also be positioned relative to the test
zones such that sample added to the sample addition zone at the
same time that the buffer is added to the buffer addition zone. The
sample addition zone may also be positioned relative to the test
zones such that the sample can be added to the test strip before
the buffer is added and nevertheless, the sample still reaches the
distal diffusion point of the buffer after the distal diffusion
front of the buffer has diffused to the distal diffusion zone,
reversed direction and begun diffusing in a proximal direction.
[0014] According to any of the above test strip embodiments, 1, 2,
3 or more test zones may be control zones with one or more control
binding agents immobilized therein. The control zones may be used
to calibrate the test strip, may be used to confirm whether or not
the test strip performed as intended, may be used detect whether
too little or too much buffer was added and may be used to detect
whether too little sample was added.
[0015] In one embodiment, the test strip comprises at least a first
control zone with a control binding agent immobilized therein.
Optionally, the test zones further includes a second control zone
with a same control binding agent immobilized therein as the first
control zone. The first control zone may contain the same or a
different amount of the control binding agent than the second
control zone. In a preferred embodiment, the first control zone
contains about the same amount of the control binding agent as the
second control zone.
[0016] Also according to any of the above test strip embodiments, a
second analyte binding agent which is capable of binding to the
analyte and diffusing to the one or more test zones may be included
on the test strip. The second analyte binding agent is preferably
incorporated on the test strip adjacent either the sample addition
zone or the buffer addition zone, more preferably proximal relative
to the sample addition zone or distal relative to the buffer
addition zone such that addition of the sample or buffer causes the
second analyte binding agent to be carried with the sample or
buffer to the test zones.
[0017] The second analyte binding agent may also be delivered to
the test strip via the buffer or the sample, most preferably the
sample. The second analyte binding agent may bind to components in
the sample in addition to the analyte. Alternatively, the second
analyte binding agent may be an agent which does not bind to
components in the sample other than the analyte.
[0018] In order to facilitate detection, the second analyte binding
agent is preferably labeled with a detectable marker. As discussed
herein, any of a wide range of detectable markers known in the art
may be used. In a preferred embodiment, the second analyte binding
agent is attached to a particle which is capable of diffusing to
the one or more test zones. The particle may serve as the
detectable marker or may itself be labeled with a detectable
marker.
[0019] Also according to any of the above test strip embodiments,
the test strip may be for a competitive assay, in which case, the
test strip may include a competitive agent. The competitive agent
may compete with the analyte for binding to the first analyte
binding agent.
[0020] The competitive agent is preferably incorporated on the test
strip adjacent the sample addition zone, more preferably proximal
relative to the sample addition zone such that addition of the
sample causes the competitive agent to be carried with the sample
to the test zone.
[0021] Methods are also provided for detecting an analyte in a
sample.
[0022] In one embodiment, the method comprises delivering a buffer
to a test strip which causes a distal diffusion front of the buffer
to (a) diffuse in a distal direction to one or more test zones, at
least one of the test zones including a first analyte binding agent
immobilized therein which binds to analyte in the sample, (b)
diffuse to a terminal buffer flow zone distal to the one or more
test zones, change direction and (c) diffuse to a position proximal
to the one or more test zones; delivering a sample to the test
strip at a position distal to the terminal buffer flow zone,
delivery of the sample causing analyte in the sample to diffuse
proximally past the terminal buffer flow zone to the one or more
test zones after the distal diffusion front of the buffer diffuses
proximal to the one or more test zones, the analyte binding to the
first analyte binding agent and becoming immobilized in the test
zones; and detecting the analyte immobilized in the test zones.
[0023] According to the method, a second analyte binding agent may
be present which binds to the analyte. The second analyte binding
agent may be used to detect the immobilized analyte. The second
analyte binding agent may be contained on the test strip where the
sample is delivered, delivery of the sample causing the diffusion
of the second analyte binding agent. Alternatively, the second
analyte binding agent may be contained on the test strip proximal
to where the sample is delivered, delivery of the sample causing
the diffusion of the second analyte binding agent. Delivering the
sample to the test strip may also include delivering the second
analyte binding agent to the test strip within the sample.
[0024] In another embodiment, the method is for a competitive
assay. According to this method, a buffer is delivered to a test
strip which causes a distal diffusion front of the buffer to (a)
diffuse in a distal direction to one or more test zones, at least
one of the test zones including a first analyte binding agent
immobilized therein which binds to analyte in the sample, (b)
diffuse to a terminal buffer flow zone distal to the one or more
test zones, change direction and (c) diffuse to a position proximal
to the one or more test zones. A sample is also delivered to the
test strip at a position distal to the terminal buffer flow zone
such that delivery of the sample causes the sample diffuse
proximally past the terminal buffer flow zone to the one or more
test zones after the distal diffusion front of the buffer diffuses
proximal to the one or more test zones.
[0025] Delivery of a sample to the test strip also causes a
competitive agent to diffuse with the sample to the test zone. The
competitive agent competes with the analyte for binding to the
first analyte binding agent. The competitive agent is preferably
incorporated on the test strip adjacent the sample addition zone,
more preferably proximal relative to the sample addition zone such
that addition of the sample causes the competitive agent to be
carried with the sample to the test zone.
[0026] The method further comprises detecting the competitive agent
immobilized in the test zones. In order to facilitate detection,
the competitive agent is preferably labeled with a detectable
marker.
[0027] According to any of the method embodiments, the buffer may
be added to the test strip at a same time as the sample is added to
the test strip, before the sample is added to the test strip, or
after the sample is added to the test strip. When the sample is
added to the test strip relative to the conjugate buffer depends on
the time required for the buffer to reach the terminal buffer flow
zone which, in turn, depends on the flow design of the test
strip.
[0028] According to any of the above methods, the test zones may
include a first control zone with a control binding agent
immobilized therein, delivering the buffer causing a control agent
to diffuse distally to the first control zone and bind to the
control binding agent immobilized therein. Alternatively, the test
zones may include first and second control zones which each include
an approximately the same or significantly different amount of a
control binding agent immobilized therein, delivering the buffer
causing a control agent to diffuse distally to the first and second
control zones and bind to the control binding agent immobilized
therein.
[0029] When one or more control zones are employed, a control agent
may be contained on the test strip where the buffer is delivered,
delivery of the buffer causing the diffusion of the control agent.
Alternatively, a control agent may be contained on the test strip
distal to where the buffer is delivered, delivery of the buffer
causing the diffusion of the control agent. Delivering the buffer
to the test strip may also include delivering the control agent to
the test strip -within the buffer. Incorporating the control agent
into the buffer is advantageous because variability in the movement
of control agents strip to strip arising from differences in the
way in which the control agents becomes resolubilized when buffer
is added is avoided.
[0030] Also according to the above methods, detecting the second
analyte binding agent may be facilitated by labeling the second
analyte binding agent with a detectable marker, detecting the
second analyte binding agent including detecting the detectable
marker. The second analyte binding agent may be attached to a
particle. Detecting the second analyte binding agent may include
detecting the particle.
[0031] According to any of the above embodiments, the buffer
delivered to the test strip is preferably within a predetermined
volume range that the test strip has been designed to process. The
predetermined volume range is preferably between about 10 and 250
L, preferably between about 20 and 200 L, more preferably between
about 20 and 100 L, and most preferably between about 40 and 60 L.
When a buffer is delivered to the test strip within the
predetermined volume range, the terminal sample flow zone may be
designed to have a short length from a proximal end to a distal
end. For example, when a buffer is delivered to the test strip
within a range of about 35 and 45 L, the terminal flow zone may
have a length from a proximal end to a distal end of between about
I and 25 mm, more preferably 2 and 15 mm, and most preferably 3 and
10 mm.
[0032] Also according to any of the above embodiments, the first
analyte binding agent preferably does not bind to components in the
sample other than the analyte. Types of molecules that can serve as
first analyte binding agents include, but are not limited to
antibodies, engineered proteins, peptides, haptens, lysates
containing heterogeneous mixtures of antigens having analyte
binding sites, ligands and receptors. In one particular embodiment,
the first analyte binding agent is an antibody or fragment
thereof.
[0033] Also according to any of the above embodiments, the buffer
added to the buffer addition zone may comprise the sample being
tested. Optionally, the buffer may be the sample. When sample forms
all or a portion of the buffer that is added to buffer addition
zone, the buffer still performs the function of prewetting the test
strip. The ability to use sample, in whole or in part, as the
buffer allows the present invention to more easily accommodate a
wider range of sample and external liquid control matrices (e.g.,
serum, plasma, euglobulin). In addition, differences in flow
behavior within the test strip between sample and buffer can be
reduced by adding the same composition (e.g., the sample) to both
the sample and buffer addition zones.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 illustrates a top-down view of an embodiment of a
lateral flow test strip according to the present invention.
[0035] FIGS. 2A-2H illustrate a method of operation for a lateral
flow test strip according to the present invention.
[0036] FIG. 2A illustrates a buffer being added to the test
strip.
[0037] FIG. 2B illustrates the buffer flowing within the test
strip.
[0038] FIG. 2C illustrates the test strip when the buffer has
flowed a distance within the test strip in the direction opposite
an absorbent zone to within a terminal buffer flow zone.
[0039] FIG. 2D illustrates the test strip where the buffer is
flowing back toward the absorbent zone.
[0040] FIG. 2E illustrates the addition of a sample to the test
strip.
[0041] FIG. 2F illustrates the flow of the sample within the test
strip toward the absorbent zone.
[0042] FIG. 2G illustrates the flow of the sample within the test
strip past the test zone.
[0043] FIG. 2H illustrates the flow of the sample within the test
strip into the absorbent zone.
[0044] FIGS. 3A-3H a method of operation for a lateral flow test
strip according to the present invention.
[0045] FIG. 3A illustrates a sample and buffer being added to the
test strip.
[0046] FIG. 3B illustrates the sample and buffer flowing within the
test strip.
[0047] FIG. 3C illustrates the test strip when the buffer has
flowed a distance within the test strip in the direction opposite
an absorbent zone to a to within terminal buffer flow zone.
[0048] FIG. 3D illustrates the test strip where the buffer is
flowing back toward the absorbent zone.
[0049] FIG. 3E illustrates the sample continuing to flow toward the
buffer flow.
[0050] FIG. 3F illustrates the sample having flowed past the
terminal buffer flow zone.
[0051] FIG. 3G illustrates the flow of the sample within the test
strip past the test zone.
[0052] FIG. 3H illustrates the flow of the sample within the test
strip into the absorbent zone.
[0053] FIG. 4 illustrates a test strip design where the sample
addition zone is positioned adjacent the buffer addition zone.
[0054] FIGS. 5A-5C illustrate various cartridge designs into which
a test strip according to the present invention can be
positioned.
[0055] FIG. 5A illustrates a cartridge design adapted for the test
strip illustrated in FIGS. 2A-2H.
[0056] FIG. 5B illustrates a cartridge design adapted for the test
strip illustrated in FIGS. 3A-3H where the buffer addition zone is
positioned an extended distance from the sample addition zone such
that the sample and wash buffer can be added at the same time.
[0057] FIG. 5C illustrates a cartridge design adapted for the test
strip illustrated in FIG. 4 where the sample addition zone is
positioned adjacent the buffer addition zone, the test zone being
positioned an extended distance from the sample addition zone.
[0058] FIG. 6A illustrates the layout of a FLEXPACKJHP test strip
manufactured by Abbott.
[0059] FIG. 6B illustrates the operation of the test strip
illustrated in FIG. 6A.
[0060] FIG. 7 illustrates a side break-away view of the lateral
flow test strip illustrated in FIG. 1.
[0061] FIG. 8 illustrates the results from the TSH assay performed
in Example 2.
[0062] FIG. 9 shows a standard curve derived from the TSH assay
results shown in FIG. 8.
[0063] FIG. 10 illustrates the results from the PSA assay performed
in Example 3.
[0064] FIG. 11 shows a standard curve derived from the PSA assay
results shown in FIG. 10.
[0065] FIG. 12 illustrates a comparison between the performance of
the ReLIA.TM. TSH assay when sample is added to both the top and
bottom ports and when sample is added only to the bottom port.
[0066] FIG. 13 illustrates the reproducible of measuring TSH levels
using the test strip of example 4.
[0067] FIG. 14 illustrate TSH values relative to a standard curve
for the test strip of example 4.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0068] The present invention relates to lateral flow test strips
and methods for employing such test strips which exhibit greater
precision and accuracy. More specifically, the lateral flow test
strips and methods of the present invention reduce performance
variability, most likely due to interferences that might affect the
absolute amount of binding of either analyte binding agent or
control binding agent to a test zone, caused by variations in
liquid flow rates across the test strip.
[0069] The present invention addresses the problem that the flow
rate of a wet test strip is significantly different than the flow
rate of a dry test strip. For example, fluid tends to flow faster
when the test strip is dry than when it is wet. In order to
minimize these flow rate influences, the present invention provides
test strips which are designed to be prewet prior to the addition
of a sample, thereby equilibrating the flow rate of the test strip
so that a sample, once added, moves through the test strip at a
more uniform rate across the test strip. In one embodiment, the
test strip is prewet using the same sample that is being tested. By
using the same sample as both a prewetting solution and as a
sample, flow rate differences are further minimized.
[0070] Given that test strips need to yield reliable and consistent
results independent of the person using the test strips, an
important aspect of the present invention is the simplicity with
which a test strip may be prewet to afford more uniform sample
velocity. As will be discussed herein in greater detail, the design
of the test strips of the present invention cause a prewetting
solution, referred to herein as a buffer, to flow across the one or
more test zones and control zones and then independently flow back
toward the buffer addition zone without unintended portions of the
strip becoming wet. This controlled flow and prewetting of the test
strip accomplishes the desired results of providing a test strip
that may be used with consistency, reproducibility and eliminates
the need for operator intervention.
[0071] A further feature of the test strips of the present
invention is the reduced timing sensitivity of the test strips
regarding when buffer and sample is added to the test strip.
Instead, the test strips of the present invention allow sample to
be added within a broader time window after buffer is added.
[0072] Other factors influencing lateral flow test results include:
1) variability in the release of an analyte binding agent or the
control agent from a conjugate pad, 2) device to device variation
in the non-specific binding of the analyte binding population to
the test strip, 3) variability in the movement of the analyte
binding population through or along the test strip during the assay
due to variation in the pore size of the test strip or membrane
strip materials or non-specific aggregation of the analyte binding
agent. These other sources of variability are also reduced by the
test strips of the present invention.
[0073] According to one embodiment, a test strip is provided which
comprises a buffer addition zone to which a buffer is added to
prewet the test strip; an absorbent zone proximal to the buffer
addition zone; one or more test zones distal to the buffer addition
zone, at least one of the test zones including a first analyte
binding agent immobilized therein which is capable of binding to
the analyte to be detected; and a terminal buffer flow zone distal
to the one or more test zones. The absorbent zone is positioned
relative to the buffer addition zone and has an absorption capacity
relative to the other zones of the test strip such that a distal
diffusion front of a buffer added to the buffer addition zone
diffuses from the buffer addition zone to a distal diffusion point
within the terminal buffer flow zone and then reverses direction,
independent of any user intervention, and diffuses proximal
relative to the one or more test zones.
[0074] The independent flowing back of the buffer toward the buffer
addition zone is achieved by positioning an absorbent zone relative
to the buffer addition zone such that when a volume of buffer
(within a predetermined volume range for that test strip) is added
to the test strip, the diffusion front of the buffer expands across
the one or more test zones to a terminal buffer flow zone. When the
buffer reaches the terminal buffer flow zone, the absorbent
properties of the absorbent zone causes the buffer to be drawn
backward across the test zones toward the buffer addition zone and
ultimately into the absorbent zone.
[0075] By causing buffer to flow across the one or more test zones
and then independently flow back toward the buffer addition zone,
the test strip is effectively prewet prior to the addition of
sample. As a result, when sample is added to the test strip, the
sample is believed to flow within the test strip at a more
consistent velocity, thereby yielding more consistent results.
[0076] The ability to cause the buffer to flow back toward the
buffer addition zone independent of any user interaction reduces
the time criticality of when sample is added to the test strip. As
will be discussed herein in greater detail, the self-timing
features of test strips according to the present invention provides
several significant advantages over previous test strips.
[0077] The test strip also comprises a sample addition zone that is
distal to the terminal buffer flow zone. When a sample is added to
the sample addition zone, the sample diffuses within the test strip
in a proximal direction across the terminal buffer flow zone,
across the one or more test zones, and ultimately to the absorbent
zone. Analyte in the sample binds to one or more test zones and is
detected there.
[0078] FIG. 1 illustrates a top-down view of an embodiment of a
lateral flow test strip 100 according to the present invention. As
illustrated, the test strip 100 has proximal and distal ends 102,
104 respectively and can be divided into several different zones.
The test strip includes a buffer addition zone 106 where a buffer
may be added to the test strip 100. An absorbent zone 108 is
positioned proximal to the buffer addition zone 106. One or more
test zones 110, 112, 114 are positioned distal to the buffer
addition zone 106. The test strip 100 also includes a terminal
buffer flow zone 116 distal to the one or more test zones 110, 112,
114. Each of the above mentioned zones are in fluid diffusion
communication with each other.
[0079] As illustrated, the test strip also includes a sample
addition zone 118 distal to the terminal buffer flow zone 116. The
sample addition zone 118 may be a zone where sample may be added to
the test strip. Alternatively, the sample addition zone 118 may
simply correspond to a zone to which sample diffuses from a more
distal point on the test strip.
[0080] The test strip may also include a zone distal to the
terminal buffer flow zone 116 which includes either a second
analyte binding agent in the case of a direct assay or a
competitive agent in the case of a competitive assay. In FIG. 1,
the sample addition zone 118 may serve as the zone comprising the
second analyte binding agent or the competitive agent.
Alternatively, the zone comprising the second analyte binding agent
or the competitive agent may be proximal to the sample addition
zone 118.
[0081] It is noted that the layout of the test strip illustrated in
FIG. 1 is linear in design. However, non-linear layouts, such as
the layout illustrated in FIG. 4, are also intended for the test
strips according to the present invention.
[0082] FIGS. 2A-2H illustrate a method of operation of a lateral
flow test strip, such as the one illustrated in FIG. 1. Prior to
performing an assay using a test strip according to the present
invention, a fluid sample is obtained that is believed to contain
the analyte to be detected. The sample can include any fluid that
wets the test strip and has a viscosity that is sufficient to allow
movement of the sample across the test strip. In a preferred
embodiment, the sample is an aqueous solution (such as a bodily
fluid).
[0083] Also prior to performing an assay, buffer is obtained which
is to be added to the test strip. As described herein, the buffer
may optionally contain a control agent. Incorporating the control
agent into the buffer is advantageous because variability in the
movement of control agents strip to strip arising from differences
in the way in which the control agents becomes resolubilized when
buffer is added is avoided. As also described herein, the buffer
added to the buffer addition zone may comprise the sample being
tested. Optionally, the buffer may be the sample. When sample forms
all or a portion of the buffer that is added to buffer addition
zone, the buffer still performs the function of prewetting the test
strip. The ability to use sample, in whole or in part, as the
buffer allows the present invention to more easily accommodate a
wider range of sample and external liquid control matrices (serum,
plasma, euglobulin). In addition, differences in flow behavior
within the test strip between sample and buffer can be reduced by
adding the same composition (e.g., the sample) to both the sample
and buffer addition zones.
[0084] FIG. 2A illustrates buffer 120 being added to the buffer
addition zone 106 of the test strip 100. It is noted that the test
strip is designed for use with a volume of buffer that is within a
particular volume range. More specifically, delivering buffer to
the buffer addition zone within the predetermined volume range
causes the buffer to diffuse distally beyond the test zones into
the terminal buffer flow zone 116, but not beyond the terminal
buffer flow zone 116 (as illustrated in FIG. 2D).
[0085] As illustrated in FIG. 2B, the buffer 120 begins to diffuse
both proximally and distally across the test strip after being
added to the test strip. As illustrated in FIG. 2C, the distal
front 124 of the buffer 120 diffuses across the one or more test
zones 110, 112, 114 to within the terminal buffer flow zone 116. As
illustrated in FIG. 2D, the distal front 124 of the buffer 120
ultimately extends to a point within the terminal buffer flow zone
116.
[0086] When the volume of the buffer added to the test strip is
within a predetermined volume range for which the test strip is
designed, the distal front 124 of the buffer 120 reaches a distal
diffusion point corresponding to a point of maximum distal flow
somewhere within the terminal buffer flow zone 116. At this point,
as illustrated in FIG. 2E, capillary action by the absorbent zone
108 draws the buffer proximally toward the absorbent zone 108. As
the buffer is drawn into the absorbent zone 108, the distal front
124 of the buffer recedes proximally.
[0087] As can be seen from FIGS. 2A-2D, a feature of the present
invention is the control of where and how the buffer flows within
the test strip. The buffer delivered to the test strip is
preferably within a predetermined volume range that the test strip
has been designed to process. The predetermined volume range is
preferably between about 10 and 250 L, preferably between about 20
and 200 L, more preferably between about 20 and 100 L, and most
preferably between about 40 and 60 L. When buffer is delivered to a
test strip within these ranges, the flow of the buffer stops within
the terminal buffer flow zone.
[0088] The terminal buffer flow zone may be designed to have a
short length from a proximal end to a distal end. For example, when
buffer is delivered to the test strip within a range of about 35
and 45 L, the terminal buffer flow zone may have a length from a
proximal end to a distal end of between about 1 and 25 mm, more
preferably 2 and 15 mm, and most preferably 3 and 10 mm.
[0089] Positioned within one of the test zones (e.g., test zone
112) is a first analyte binding agent which binds to an analyte in
a sample which the test strip is designed to detect. Analyte
present in the portion of the sample which flows across the test
zones is immobilized in test zone 112 by the first analyte binding
agent.
[0090] FIG. 2E illustrates the addition of a sample 122 to the test
strip at the sample addition zone 118 after the buffer has reached
the terminal buffer flow zone. The volume of sample added is
preferably between about 10 and 250 L, preferably between about 20
and 150 L, more preferably between about 50 and 150 L, and most
preferably between about 75 and 125 L. It is noted that the most
preferred volume of sample to add to a test strip will vary
depending on the assay.
[0091] The sample 122 may contain one or more different second
analyte binding agents which can bind to the analyte and enable
analyte immobilized in the test zones to be detected. It is noted
that the sample addition zone 118 may optionally include the one or
more second analyte binding agents used to detect immobilized
analyte. In that instance, addition of the sample 122 serves to
initiate diffusion of the one or more second analyte binding agents
across the test zones.
[0092] As illustrated in FIGS. 2F and 2G, the sample 122 flows
proximally across the test strip toward the absorbent zone 108,
thereby causing both analytes in the sample and the one or more
second analyte binding agents to move across the test zones 110,
112, 114 and bind to immobilized analyte.
[0093] As illustrated in FIG. 2H, capillary action by the absorbent
zone 108 causes the buffer 120 to diffuse into the absorbent zone
108. Meanwhile, the sample 122 continues to diffuse proximally
across the test zones 110, 112, 114 and into the absorbent zone
108. Any of the one or more second analyte binding agents that were
not immobilized in the test zones 110, 112, 114 are carried with
the sample 122 into the absorbent zone 108.
[0094] In regard to the embodiment illustrated in FIGS. 2A-2H, it
is noted that the sample 122 should be added to the test strip
after the buffer 120 has reached the test zones 110, 112, 114 and
preferably after the buffer has reached the terminal buffer flow
zone 116 and has begun to diffuse back toward the absorbent zone
108. This allows the buffer 120 to prewet the test strip.
[0095] FIGS. 3A-3H illustrate an alternative test strip design and
method of operation for the test strip. In this embodiment, the
buffer and sample are added at the same time. In order for the
buffer and sample to be added at about the same time, it is
necessary for the sample to reach the test zones 210, 212, 214
after the buffer has contacted the test zones. It is preferred that
the sample reach the test zones after the buffer has begun
diffusing back across the test zones toward the absorbent zone
208.
[0096] Delaying when the sample reaches the test zones is
accomplished in this embodiment by creating a longer distance
between sample addition zone 218 and the terminal buffer flow zone
216 as compared to the test strip design illustrated in FIGS.
2A-2H. Alternatively, one can use a material which causes the
sample to diffuse at a slower rate.
[0097] FIG. 3A illustrates a buffer 220 being added to a buffer
addition zone 206 of the test strip 200. Meanwhile, a sample 222 is
added to a sample addition zone 218 at about the same time that the
buffer is added to the test strip.
[0098] As illustrated in FIG. 3B, the buffer 220 begins to diffuse
both proximally and distally within the test strip once added to
the test strip. Meanwhile, the sample 222 also diffuses proximally
and optionally distally within the test strip.
[0099] As illustrated in FIG. 3C, the distal front 224 of the
buffer 220 diffuses across one or more test zones 210, 212, 214 to
within a terminal buffer flow zone 216. Meanwhile, the sample 222
continues to diffuse proximally within the test strip toward the
test zones.
[0100] As illustrated in FIG. 3D, the distal front 224 of the
buffer 220 ultimately extends to a point within the terminal buffer
flow zone 216. At the time when the buffer is in the terminal
buffer flow zone 216, the sample 222 has not yet reached that
zone.
[0101] As illustrated in FIG. 3E, capillary action by the absorbent
zone 208 draws the buffer proximally toward the absorbent zone 208.
As the buffer is drawn into the absorbent zone 208, the distal
front 224 of the buffer flows proximally.
[0102] FIG. 3F illustrates the sample 222 reaching the test zones.
As can be seen, by the time the sample 222 reaches the test zones,
the distal front 224 of the buffer has already flowed proximally
out of the terminal sample flow zone 216 and the test zones 210,
212, 214. Positioned within one of the test zones (e.g., test zone
212) is a first analyte binding agent which binds to analyte in the
sample which the test strip is designed to detect. Analyte present
in the portion of the sample which flows across the test zones is
immobilized in test zone 212 by the first analyte binding
agent.
[0103] As illustrated in FIGS. 3G and 3H, capillary action by the
absorbent zone 208 causes the buffer to withdraw into the absorbent
zone 208. Meanwhile, the sample 222 continues to diffuse proximally
across the test zones 210, 212, 214 and into the absorbent zone
208. Any of the one or more second analyte binding agents that were
not immobilized in the test zones 210, 212, 214 are carried with
the sample 222 into the absorbent zone 208.
[0104] The sample 222 added to the test strip may contain one or
more second analyte binding agents which can bind to the analyte
and enable analyte immobilized in the test zones to be detected.
Alternatively, the test strip may include a conjugate zone distal
to the terminal buffer flow zone 216 which contains one or more
second analyte binding agents. The sample addition zone 218 may
also serve as the conjugate zone. When the one or more second
analyte binding agents are preloaded onto the test strip, the
sample 222 serves to initiate diffusion of the one or more second
analyte binding agents across the test zones toward the absorbent
zone.
[0105] As illustrated in FIGS. 3A-3H, the sample may be added to
the test strip before the buffer reaches the test zones by
designing the diffusion path of the test strip such that the sample
does not reach the test zones until after the buffer has diffused
over and then back from the test zones. It is noted that the
diffusion of the sample to the test zones may be sufficiently
delayed that one adds the sample to the test strip prior to adding
the buffer to the test strip.
[0106] In regard to the embodiments illustrated in FIGS. 2A-2H and
3 A-3H, it is noted that the method is a direct assay, i.e., the
amount of analyte present is measured by measuring the amount of
analyte immobilized in a test zone. Competitive assays, i.e.,
assays where the amount of analyte present is measured by measuring
how much less of a competitive agent is immobilized in a test zone.
In order to perform a competitive assay, the operation of the test
strips illustrated in FIGS. 2A-2H and 3A-3H need only be modified
by employing a competitive agent which competes with the analyte to
bind to the first analyte binding agent.
[0107] FIG. 4 illustrates an alternative test strip design for a
lateral flow test strip according to the present invention. The
operation of the test strip is similar to the operation described
in FIGS. 3A-3H. The same reference numerals are employed in FIG. 4
as in FIGS. 3A-3H. As illustrated in FIG. 4, the buffer addition
zone 206 is positioned adjacent the sample addition zone 218. This
allows for a more compact test strip design while also allowing the
sample and buffer to be added simultaneously.
[0108] One feature of the test strip design illustrated in FIG. 4
is that the sample and buffer are added to the same end of the test
strip. It is also noted that the test zones 210, 212, 214 are
positioned toward an opposite end of the sample and buffer addition
zones 206, 218. This makes it possible for the test zones to be
positioned within a sample reader while the sample and buffer
addition zones are outside the sample reader. This, in turn, allows
sample and buffer to be added to the test strip while the test
strip is in a test strip reader.
[0109] FIGS. 5A-5C illustrate various cartridge designs into which
test strips according to the present invention can be positioned.
In each cartridge design, the cartridge includes a buffer addition
port 240 adjacent the buffer addition zone 206 of the test strip.
The cartridge also includes a sample addition port 242 adjacent the
sample addition zone 218 of the test strip. The cartridge also
includes a test window 244 adjacent the test zones 210, 212, 214 of
the test strip.
[0110] FIG. 5A illustrates a cartridge design adapted for the test
strip illustrated in FIGS. 2A-2H. FIG. 5B illustrates a cartridge
design adapted for the test strip illustrated in FIGS. 3A-3H where
the buffer addition zone is positioned an extended distance from
the sample addition zone such that the sample and buffer can be
added to the test strip at about the same time. FIG. 5C illustrates
a cartridge design adapted for the test strip illustrated in FIG. 4
where the buffer addition zone is positioned adjacent the sample
addition zone, the test zone being positioned an extended distance
from the sample addition zone.
[0111] It is noted with regard to FIGS. 2-4 that a feature of the
test strips of the present invention is the test strip's inherent
ability to expose test zones on the test strip to buffer for a
period of time and then to cause the buffer to diffuse away from
the test zones prior to the sample reaching the test zones. This
feature is made possible by matching (1) the positioning of the
absorbent zone relative to the buffer addition zone with (2) the
absorbent capacity of the test strip between the buffer addition
zone and the terminal buffer flow zone and (3) the volume of the
buffer to be delivered to the test strip. If too much buffer is
delivered, the buffer will diffuse beyond the terminal buffer flow
zone. If too little buffer is delivered, the buffer does not
diffuse far enough in the test strip to reach the test zones and
thus does not adequately prewet the test strip.
[0112] The test strip's ability to expose the test zones to buffer
for a limited period of time and then cause the buffer to be
removed from the test zones confers a timing independence to the
test strip which enhances the test strip's precision and ease of
use. For example, test results are not dependent on when the sample
and buffer are added to the test strip. As a result, the test
strips need not be carefully monitored regarding when the sample
should be added. In this regard, the window of time after the
buffer has been added when sample should be added to the test strip
is substantially eliminated by the present invention.
[0113] The dynamics of using the volume of the buffer delivered to
the test strip to control how the buffer diffuses within the test
strip will now be illustrated in regard to FIG. 1. As discussed
previously, FIG. 1 illustrates a test strip which has proximal and
distal ends 102, 104 respectively and is divided into several
distinct zones. The test strip includes a buffer addition zone 106
where a buffer is added to the test strip. An absorbent zone 108 is
positioned proximal to the buffer addition zone 106. A test zone
112 is positioned distal to the buffer addition zone 106. A
terminal buffer flow zone 116 is positioned distal to the test zone
112. A sample addition zone 118 is positioned distal to the
terminal buffer flow zone 116.
[0114] For the purpose of illustration, assume that the test zone
112 includes a first analyte binding agent and the sample addition
zone 118 includes a second analyte binding agent labeled with a
detectable marker. Also assume that the test strip is designed such
that a buffer volume of 30 L will cause the buffer to diffuse to
but not beyond the test zone 112. Meanwhile, a buffer volume of 50
L will cause the buffer to diffuse to the distal end of the
terminal buffer flow zone 116.
[0115] If buffer is delivered to the test strip within the 30-50 L
volume range, the distal front of the buffer will diffuse past the
test zone 112. Distal advancement of the buffer will stop within
the terminal buffer flow zone 116. The buffer then flows back in
the proximal direction toward the absorbent zone 108 past the test
zone 112, thereby prewetting the test strip. When the sample is
added, the sample causes the analyte in the sample and the second
analyte binding agent to diffuse across the test zone 112. The
second analyte binding agent binds to the analyte which in turn
binds to the first analyte binding agent immobilized in the test
zone 112. Other components in the sample will not bind to the first
analyte binding agent antibody since the first analyte binding
agent is selective for the analyte. Since the buffer diffuses away
from the test zone 112 prior to the sample reaching the test zone
112, the prior addition of the buffer prewets the test strip but
the flow of the buffer does not interfere with the flow of the
sample within the test strip.
[0116] If a buffer volume of less than 30 L is delivered (e.g., 25
L) to the test strip, the buffer never diffuses to the test zone
112. As a result, the buffer does not prewet the test strip in the
test zone 112. When the sample is added, the sample has to flow
across a combination of dry test strip and wet test strip which can
create variations due to differences in flow rates.
[0117] If the buffer volume delivered is greater than 50 L (e.g.,
55 L), the buffer will diffuse past the test zone 112 and past the
terminal buffer flow zone 116 into the sample addition zone. When
too much buffer is added, the test strip could be flooded, thereby
interfering with the test strip's operation. Also, in some
embodiments, the buffer could cause diffusion of a second analyte
binding agent or a competitive agent positioned distal relative to
the terminal buffer flow zone 116.
[0118] As has been described above, one of the advantages of the
test strips of the present invention is their self-timing property.
In order to explain the significance of these properties, a
comparison will now be made to the FLEXPACKJHP test strip
manufactured by Abbott which is illustrated in FIGS. 6A and 6B.
[0119] FIG. 6A illustrates the layout of the test strip. As
illustrated, the test strip includes two separate sections 310, 312
which are attached to each other by a hinge 314. Section 310 on the
right includes a test strip 316 which includes a sample addition
zone 318, a test zone 319, a limit line 320, and a conjugate buffer
transfer pad 322. Section 312 on the left includes an absorbent pad
324 which is positioned opposite the sample addition zone 318, a
conjugate buffer addition pad 326 which is positioned opposite the
conjugate buffer transfer pad 322, and a test window 328 which is
positioned opposite the test zone 319. The opposing positionings of
the absorbent pad 324, the conjugate buffer addition pad 326, and
the test window 328 allows the absorbent pad 324 to contact the
sample addition zone 318 and the conjugate buffer addition pad 326
to contact the conjugate buffer transfer pad 322 when the first and
second sections 310, 312 are brought into contact with each other.
In addition, the test zone 319 can be seen through the test window
328 when the first and second sections 310, 312 are brought
together.
[0120] FIG. 6B illustrates the operation of the test strip
illustrated in FIG. 6A. As illustrated, a conjugate buffer 330 is
added to the conjugate buffer addition pad 326. The conjugate
buffer addition pad 326 includes a second analyte binding agent
(e.g., an antibody) capable of binding to an analyte in the sample
to be detected. The second analyte binding agent is labeled with a
detectable marker which allows the second analyte binding agent to
be visualized. The second analyte binding agent is not specific for
the analyte and thus can bind to other components in the
sample.
[0121] A sample 332 is then taken and added to the sample addition
zone 318. Once added, the sample diffuses through the test strip
316 from the sample addition zone 318 across the test zone 319. The
test zone 319 includes an immobilized first analyte binding agent
(e.g., an antibody) which selectively binds to an analyte in the
sample which the test strip is designed to detect. When the sample
traverses the test zone 319, analyte in the sample binds to the
first analyte binding agent and is immobilized in the test zone
319.
[0122] When the diffusion front of the sample reaches the limit
line 320, the user is supposed to bring the first and second
sections 310, 312 together. Bringing the first and second sections
310, 312 together causes the absorbent pad 326 to draw the sample
back toward the sample addition zone 318. Meanwhile, conjugate
buffer is transferred to the conjugate buffer transfer pad 322 from
the conjugate buffer addition pad 320. The conjugate buffer
diffuses from the conjugate buffer transfer pad 322 across the test
zone 319. Second analyte binding agent that was stored in the
conjugate buffer addition zone 318 diffuses with the conjugate
buffer and contacts immobilized analyte in the test zone 319.
Observation of the visually detectable marker on the second
analyte, binding agent once immobilized in the test zone 319, is
used to detect the analyte.
[0123] As can be seen from the above description of the operation
of the FLEXPACKJHP test strip, it is necessary to determine when
the sample reaches the limit line 320 before causing the conjugate
buffer to be transferred from the buffer addition zone 318 to the
conjugate buffer addition pad 320 and begin flowing toward the test
zone 319. It is also necessary to take the affirmative step of
contacting the sample addition zone 318 with the absorbent pad 324
in order to cause the sample to be withdrawn from the test zone
319.
[0124] The design of the test strips of the present invention, for
example those illustrated in FIGS. 2-4, eliminate the need to
monitor the test strip to determine when to begin the removal of
the sample from the test zone. It is noted that no monitoring is
required and that the sample is added after buffer in the test
strips of the present invention as opposed to the FLEXPACKJHP test
strip.
[0125] In addition, since the buffer withdraws automatically, one
need not carefully monitor the test strip regarding when to add the
sample. Rather, test results using the test strips of the present
invention are not dependent on when the sample reaches the test
zones after the buffer diffuses from the test zones.
[0126] Lateral flow assays according to the invention may find use
in a variety of applications. For example, the assays may be used
to assay for human diseases, such as infectious diseases, or any
other human diseases involving recognizable epitopes (e.g. cancer,
autoimmune disease, cardiovascular conditions, hormone testing, and
pathology). The assays may also be used in veterinary, food
testing, agricultural, or fine chemical applications. The lateral
flow assays according to the invention may be performed in variety
of ways, including use of a lateral flow assay testing apparatus,
such as that disclosed in the application Ser. No. 09/199,255,
filed Nov. 23, 1998 which is incorporated herein by reference. In a
preferable embodiment, the lateral flow assay testing apparatus
comprises a ReLIAJ testing apparatus, available from PraxSys
BioSystems (San Ramon, Calif.).
[0127] 1. Construction of Test Strips According To The Present
Invention
[0128] Methods and materials for constructing test strips according
to the present invention will now be discussed in greater detail.
It is noted that the particular construction of the test strip may
be varied, depending on the particular assay that the test strip is
intended to perform. Variations in the way in which the test strips
may be constructed beyond this example are intended to fall within
the scope of the invention.
[0129] FIG. 7 illustrates a side break-away view of the lateral
flow test strip illustrated in FIG. 1. As illustrated in FIG. 7,
the test strip 100 may include a backing strip 402 which runs a
length of the test strip. A membrane strip 404 is positioned over
the backing strip 402 and serves as a diffusion passageway for the
test strip. An absorbent pad 408 is positioned over the membrane
strip 404 within the absorbent zone 108 which is positioned toward
a proximal end of the test strip. A buffer pad 406 is positioned
over the membrane strip 404 distal to the absorbent pad 408. An
adhesive 409 may be used to attach the buffer pad 406 to the
membrane strip 404. One or more test zones 410, 412, 414 may be
formed in the membrane strip 404 distal to the sample pad 406. Some
of these test zones may be control zones and some may be for
measuring an analyte in the sample. A conjugate pad 416 is
positioned over the membrane strip 404 distal to the test zones
410, 412, 414 and distal to the terminal buffer flow zone 116. A
sample pad is positioned over or distal to the conjugate pad. A
protective cover 418 optionally may be positioned over the test
zones.
[0130] The backing strip may be made of any stable, non-porous
material that is sufficiently strong to support the materials and
strips coupled to it. Since many assays employ water as a diffusion
medium, the backing strip is preferably substantially impervious to
water. In a preferred embodiment, the backing strip is made of a
polymer film, more preferably a polyvinyl film.
[0131] The membrane strip may be made of any substance which has
sufficient porosity to allow capillary action of fluid along its
surface and through its interior. The membrane strip should have
sufficient porosity to allow movement of antibody- or
antigen-coated particles. The membrane strip should also be
wettable by the fluid used in the sample which contains the analyte
to be detected (e.g., hydrophilicity for aqueous fluids,
hydrophobicity for organic solvents). Hydrophobicity of a membrane
can be altered to render the membrane hydrophilic for use with
aqueous fluid, by processes such as those described in U.S. Pat.
No. 4,340,482, or U.S. Pat. No. 4,618,533, which describe
transformation of a hydrophobic surface into a hydrophilic surface.
Examples of substances which can be used to form a membrane strip
include: cellulose, nitrocellulose, cellulose acetate, glass fiber,
nylon, polyelectrolyte ion exchange membrane, acrylic
copolymer/nylon, and polyethersulfone. In a preferred embodiment,
the membrane strip is made of nitrocellulose.
[0132] The absorbent pad may be formed of an absorbent substance
that can absorb the fluid used as the sample and buffer. The
absorption capacity of the absorbent pad should be sufficiently
large to absorb the fluids that are delivered to the test strip.
Examples of substances suitable for use in an absorbent pad include
cellulose and glass fiber.
[0133] The sample and buffer addition pads may be formed of any
absorbent substance. Examples of substances that may be used
include cellulose, cellulose nitrate, cellulose acetate, glass
fiber, nylon, polyelectrolyte ion exchange membrane, acrylic
copolymer/nylon, and polyethersulfone.
[0134] As discussed previously, the sample addition pad may serve
as the additional role of being the conjugate pad and contain an
agent labeled with a detectable marker which is capable of binding
to the analyte to be detected in the sample. In competitive assays,
the sample addition pad may contain a competitive agent.
Alternatively, the test strip may include a conjugate pad separate
from the sample addition pad which contains an agent labeled with a
detectable marker which is capable of binding to the analyte to be
detected in the sample. In competitive assays, the conjugate pad
may contain a competitive agent. In FIG. 7, a conjugate pad is
shown as element 420 beneath the sample addition pad 416. It is
noted that the conjugate pad in FIG. 7 is positioned in the flow
path between the sample addition pad 416 and the remainder of the
test strip.
[0135] The protective cover, if used, may be formed of any material
which is impervious to water, and is preferably translucent or
transparent. The protective covering may be a single or multiple
layers. Preferable materials for use in the protective covering
include optically transmissive materials such as polyamide,
polyester, polyethylene, acrylic, glass, or similar materials. The
protective covering may be clear or not clear depending on method
of detection used. In a preferable embodiment, protective covering
is optically clear polyester.
[0136] 2. Assays For Use With Test Strips According To The Present
Invention
[0137] The test strips of the present invention are intended to be
employable with a wide variety of lateral flow assays involving two
analyte binding agents which each can bind to an analyte to be
detected. At least one of the binding agents should bind
selectively to the analyte. More specifically, one of the binding
agents should bind to the analyte and not bind to any other
components of the sample.
[0138] As used herein, the term, "analyte," is intended to refer to
any component of a sample (e.g., molecule, compound, or aggregate
thereof) which is to be detected and optionally quantitatively
determined by an assay test strip. Examples of analytes include
proteins, such as hormones and other secreted proteins, enzymes,
and cell surface proteins; glycoproteins; peptides; small
molecules; polysaccharides; antibodies (including monoclonal or
polyclonal Ab and portions thereof); nucleic acids; drugs; toxins;
viruses or virus particles; portions of a cell wall; and other
compounds possessing epitopes.
[0139] The first and second analyte binding agents may be any
agents which can bind to the analyte to be detected. A variety of
different types of molecules can be used as analyte binding agents,
including, for example, antibodies, engineered proteins, peptides,
haptens, and lysates containing heterogeneous mixtures of antigens
having analyte binding sites. P. Holliger et al., Trends in
Biotechnology 13:7-9 (1995); S. M. Chamow et al., Trends in
Biotechnology 14:52-60 (1996). If the analyte to be detected is a
ligand, a receptor which binds to the ligand can be used, and vice
versa. In one particular embodiment, the first and/or second
analyte binding agents are antibodies which bind to an immunogenic
portion of the analyte.
[0140] It is noted that at least one of the first and second
analyte binding agents should bind to the analyte and not bind to
any of the other components in the sample to be analyzed, referred
to herein as an analyte-selective binding agent. In one embodiment,
the first analyte binding agent which is immobilized in a test zone
is an analyte-selective binding agent and the second analyte
binding agent which is labeled with a detectable marker is capable
of binding non-selectively to the analyte. In another embodiment,
the first analyte binding agent which is immobilized in a test zone
is capable of binding non-selectively to the analyte and the second
analyte binding agent which is labeled with a detectable marker is
an analyte-selective binding agent. In yet another embodiment, both
the first and second analyte binding agents are analyte-selective
binding agents.
[0141] Examples of analyte-selective binding agents include
antibodies (monoclonal, polyclonal, and fragments thereof) which
have a narrow binding affinity to only a particular type of
biomolecule, such as a protein or receptor. The detectable marker
attached to the second analyte binding agent may comprise a wide
variety of materials, so long as the marker can be detected.
Examples of detectable markers include, but are not limited to
particles, luminescent labels; colorimetric labels, fluorescent
labels; chemical labels; enzymes; radioactive labels; or radio
frequency labels; metal colloids; and chemiluminescent labels.
Examples of common detection methodologies include, but are not
limited to optical methods, such as measuring light scattering,
simple reflectance, luminometer or photomultiplier tube;
radioactivity (measured with a Geiger counter, etc.); electrical
conductivity or dielectric (capacitance); electrochemical detection
of released electroactive agents, such as indium, bismuth, gallium
or tellurium ions, as described by Hayes et al. (Analytical Chem.
66:1860-1865 (1994)) or ferrocyanide as suggested by Roberts and
Durst (Analytical Chem. 67:482-491 (1995)) wherein ferrocyanide
encapsulated within a liposome is released by addition of a drop of
detergent at the detection zone with subsequent electrochemical
detection of the released ferrocyanide. Other conventional methods
may also be used, as appropriate.
[0142] It may be desired to assay two or more different analytes
using the same test strip. In such instances, it may be desirable
to employ different detectable markers on the same test strip where
each detectable marker detects a different analyte. For example,
different detectable markers may be attached to different
analyte-selective binding agents. The different detectable markers
may be different fluorescent agents which fluoresce at different
wavelengths.
[0143] When detecting two or more different analytes using the same
test strip, separate test zones may optionally be formed on the
test strip for each analyte to be detected. The same detectable
marker may be used for all of the analytes. Alternatively,
different detectable markers, as described above, may be used for
the different analytes in order to prevent one test zone being
confused with another.
[0144] In a preferable embodiment, the detectable marker is a
particle. Examples of particles that may be used include, but are
not limited to, colloidal gold particles; colloidal sulphur
particles; colloidal selenium particles; colloidal barium sulfate
particles; colloidal iron sulfate particles; metal iodate
particles; silver halide particles; silica particles; colloidal
metal (hydrous) oxide particles; colloidal metal sulfide particles;
colloidal lead selenide particles; colloidal cadmium selenide
particles; colloidal metal phosphate particles; colloidal metal
ferrite particles; any of the above-mentioned colloidal particles
coated with organic or inorganic layers; protein or peptide
molecules; liposomes; or organic polymer latex particles, such as
polystyrene latex beads.
[0145] A preferred class of particles is colloidal gold particles.
Colloidal gold particles may be made by any conventional method,
such as the methods outlined in G. Frens, 1973 Nature Physical
Science, 241:20 (1973). Alternative methods may be described in
U.S. Pat. Nos. 5,578,577, 5,141,850; 4,775,636; 4,853,335;
4,859,612; 5,079,172; 5,202,267; 5,514,602; 5,616,467;
5,681,775.
[0146] The selection of particle size may influence such factors as
stability of bulk sol reagent and its conjugates, efficiency and
completeness of release of particles from conjugate pad, speed and
completeness of the reaction. Also, particle surface area may
influence steric hindrance between bound moieties. Particle size
may also be selected based on the porosity of the membrane strip.
The particles are preferably sufficiently small to diffuse along
the membrane by capillary action of the conjugate buffer.
[0147] Particles may be labeled to facilitate detection. Examples
of labels include, but are not limited to, luminescent labels;
colorimetric labels, such as dyes; fluorescent labels; or chemical
labels, such as electroactive agents (e.g., ferrocyanide); enzymes;
radioactive labels; or radio frequency labels.
[0148] The number of particles present in the test strip may vary,
depending on the size and composition of the particles, the
composition of the test strip and membrane strip, and the level of
sensitivity of the assay. The number of particles typically ranges
between about 1.times.10.sup.9 and about 1.times.10.sup.13
particles, although fewer than about 1.times.10.sup.9 particles may
be used. In a preferred embodiment, the number of particles is
about 1.times.10.sup.11 particles.
[0149] 3. Control Test Zones
[0150] As illustrated in FIG. 1, a plurality of test zones 110,
112, 114 may be included on the test strip. Each test zone is
located such that an automatic or semi-automatic analytical
instrument, or a human reader, may determine certain results of the
lateral flow assay.
[0151] As discussed previously, immobilized in at least one of the
test zones is a first analyte binding agent which is capable of
binding to an analyte in the sample which the test strip is
designed to detect. In some embodiments, it may be desirable for
some of the other test zones to serve as one or more control zones
where one or more control binding agents have been immobilized.
Control agents capable of binding to the control binding agent may
be positioned on the test strip at various locations or added to
the test strip when the assay is being performed. The control
agents are preferably labeled with a detectable marker, such as the
detectable markers described above, to facilitate detection of the
control agent binding to the control binding agent immobilized in a
control zone.
[0152] The control agents and control binding agents may be used in
combination to perform a variety of control functions. For example,
the control binding pairs may be used to confirm whether the sample
and buffer have diff-used properly within the test strip. The
control binding pairs are also employable as internal standards and
allow analyte measurement results to be compared between different
test strips. This can be used to correct for strip-to-strip
variability. Such correction would be impractical with external
controls that are based, for example, on a statistical sampling of
strips. Additionally, lot-to-lot-and run-to-run variations between
different test strips may be minimized by the use of control
binding pairs. Furthermore, the effects of non-specific binding, as
discussed further below, may be reduced. All of these corrections
are difficult to accomplish using external, off-strip controls.
[0153] A wide variety of agents are known in the art which may be
used as a member of the control binding pair. For example, at least
one member of the control binding pair may be a naturally occurring
or engineered protein. The control binding pair may also be a
receptor-ligand pair. Additionally, at least one member of the
control binding pair may be an antigen, another organic molecule,
or a hapten conjugated to a protein non-specific for the analyte of
interest. Descriptions of other suitable members of control binding
pairs may be found in U.S. Pat. No. 5,096,837, and include IgG,
other immunoglobulins, bovine serum albumin (BSA), other albumins,
casein, and globulin.
[0154] Desirable characteristics for control agent-control binding
agent pairs include, but are not limited to stability in bulk,
non-specificity for analyte of interest, reproducibility and
predictability of performance in test, molecular size, and avidity
of binding for each other.
[0155] In a preferred embodiment, members of the control binding
pair do not bind to anything that might be present in the test
strip, e.g., from the sample. In one embodiment, the control
binding agent comprises rabbit anti-dinitrophenol (anti-DNP)
antibody and the control agent includes a dinitrophenol conjugated
to BSA (bovine serum albumin).
[0156] In one preferred embodiment, the second analyte binding
agent and the control agent are each separately bound to different
particles.
[0157] In another preferred embodiment, both the second analyte
binding agent which diffuses along the test strip and the control
agent are attached to a single species of particle. Attachment may
be by non-specific absorption or by traditional conjugate
chemistries. Alternatively, a non-covalent binding system, such as
biotin-avidin, or even an antibody specific for the second analyte
binding agent may be used to attach the analyte binding agent to
the particle. Bifunctional and multifunctional reagents may also be
used to couple to the second analyte binding agent and the control
agent to the particle.
[0158] The number of second analyte binding agents and control
agents attached to each particle can be varied, depending on what
is appropriate for a particular assay. For example, two copies of
the second analyte binding agent and one copy of the control agent
may be attached to each particle. Alternatively, one copy of the
second analyte binding agent and two copies of the control agent
may be attached to each particle. Other variations on the ratios
between second analyte binding agent: control agent: particle can
be used depending on the particular assay in which they are to be
employed, these variations being intended to fall within the scope
of the present invention.
[0159] When the test strip includes more than one control zone, the
control zones may be used to create a calibration curve against
which a wide variety of analyte measurement results may be
compared. The control zones may also be used to troubleshoot
whether the test strip operated appropriately.
[0160] In one embodiment, the test strip has at least two control
zones that have about the same concentration of control binding
agent. It is noted that it is typically easier and more economical
to deliver the same amount of control binding agent to different
control zones.
[0161] Alternatively, the concentration of control agent in one of
the control zones may be greater than the concentration of control
agent in another of the control zones. In this instance, the amount
of control binding pairs will be higher in the control zone with
the higher concentration of control binding agent than in the other
control zone.
[0162] Incorporating more than one control zone on a test strip can
be used to provide the test strip with a wider dynamic range than
conventional lateral flow assays. In preferred embodiments, test
strips with 2, 3 or more control zones are used with a relative
scale methodology that permits mapping of amounts of control
binding pairs detected onto the same scale on which amounts of
analyte detected are reported.
[0163] Incorporating more than one control zone on a test strip can
also be used to evaluate the performance of the test strip. For
example, an apparatus for evaluating an analyte in a sample can
determine a ratio between the measured amounts of control binding
agent in at least two control measurement zones, and detect whether
an error has occurred in a test strip based on whether the
determined ratio falls outside of predetermined acceptable maximum
and minimum ratio ranges for that test strip. If an error is not
deemed to have occurred, the apparatus may proceed to evaluate the
amount of analyte in the sample. If an error is deemed to possibly
have occurred, the instrument may notify an instrument operator of
the potential error.
[0164] When the concentrations of control binding agent in the
first and second control zones are about the same, the
predetermined acceptable maximum and minimum ratio may both be near
about 1. When the concentrations of control binding agent in the
frst and second control zones are different (e.g., the first
control zone has three times as much control binding agent as the
second control zone), the predetermined acceptable maximum and
minimum ratio may both be near about 3.
[0165] An apparatus used to measure the test strips may include
executable logic that determines whether the concentrations of
control binding agent in the frst and second control zones are
within a predetermined acceptable maximum and minimum ratio.
[0166] An apparatus used to measure the test strips may also
include executable logic that evaluates the amount of analyte in
the sample based on a combination of the measured amount of first
analyte bound in the analyte measurement zone and the measured
amount of control binding agent bound in the first control
measurement zone.
[0167] The apparatus may include executable logic that evaluates
the amount of analyte in the sample based on a combination of the
measured amount of first analyte bound in the analyte measurement
zone and the measured amount of control binding agent bound in the
first and second control measurement zones.
[0168] The amount of control binding pairs in a given control zone
may be mapped onto the same measurement scale on which the amount
of analyte is reported, a calibration curve may be drawn through
the values of the binding pairs in the high and low control
zones.
[0169] When more than two control zones are present, a curve may be
generated that reflects any nonlinearities present in the assay
between the amount of analyte detected and the measurement against
which the amount might be mapped. While such nonlinearities might
otherwise affect assays that assume a relatively linear
relationship, they can be corrected for using multiple control
zones. 2, 3 or more control zones may be used.
[0170] In another embodiment, a single control zone may comprise
more than one type of control agent. This may be of use in
embodiments where there are more than one population of analyte
binding agents and analyte non-specific agents coupled to a
detection agent. For example, when it is desired to assay two or
more analytes of interest on the same assay strip, two populations
of analyte binding agents and analyte non-specific agents coupled
to a detection agent may be prepared. Different detection agents
may be used for each population, allowing a distinction to be drawn
between results for the two different analytes of interest. In such
circumstances, it may be desirable to use control zones comprising
different control agents or control binding pairs.
[0171] The control zones may be located in a variety of locations
within the group of test zones. It is noted that the test zones may
be placed on various locations on the test strip, depending on the
flow design of the test strip consistent with the present
invention. In a preferred embodiment, the control zones are
adjacent the test zones used to detect analytes in the sample. In a
particularly preferred embodiment, at least one control zone is
positioned proximal to a test zone used to detect an analyte in a
sample and at least one control zone is positioned distal to that
test zone.
[0172] By positioning the analyte test zone between two control
zones, the control zones can be effectively used to confirm several
operations of the test strip. For example, the control zones can
confirm that buffer was added and that sufficient buffer was added
so that the buffer completely traversed the analyte test zone in
both directions. Development of the analyte test zone confirms that
the sample was added. By measuring a relationship between the
control zones, it is also possible to confirm that sufficient
sample was added and that the strip flowed properly.
[0173] Assays are performed using a test strip which includes one
or more control regions as part of the test regions in the same
manner as described in regard to FIGS. 2A-2H and 3A-3H. It is noted
that either the test strip or the buffer may include the control
agent which binds to the control binding agent immobilized, for
example, in test zones 110 and 114 of FIGS. 2A-2H and test zones
210 and 214 of FIGS. 3A-3H. When the buffer is added, the control
agent diffuses with the buffer and binds to the control binding
agent immobilized in the control zones. When the sample is added,
the sample serves to wash any unbound control agent away from the
control zone.
[0174] Amounts of control agents immobilized in the control zones
are detected along with the detection of amounts of second analyte
binding agent immobilized in the test zones. As noted above, it is
preferred for the control agents and the second analyte binding
agent to be labeled with a detectable marker which facilitates
their detection. The amount of detectable marker in each test zone
can be readily determined by a variety of techniques known in the
art, depending on the type of detectable markers being employed.
Common examples of detection techniques include optical methods
(light scattering, simple reflectance, luminometer or
photomultiplier tube); radioactivity; electrical conductivity;
dielectric capacitance; electrochemical detection of released
electroactive agents; as has been noted above.
[0175] Once the amount of detectable markers has been measured in
each test zone, these measurements may be used to detect and
preferably quantify the amount of analyte present, preferably by
also calibrating the test strip using the amounts of detectable
markers in the control zones. For example, when one or more control
zones are employed, the amount of control agent immobilized in one
or more of the control zones may be used to quantify the amount of
first analyte binding agent relative to one or more of the control
zones. These relative intensity measurements may then be used to
more accurately determine the number of copies of analyte present
in the measurement volume.
[0176] One feature of using multiple control zones is the ability
to create a relative scale for analyte measurements. Once the
amounts of detectable markers have been quantified, these amounts
may then be mapped onto another measurement scale. For example,
while the results from measuring the analyte may be measured based
on an absolute measurement of the analyte, the results reported may
be more meaningful in other units, such as an intensity relative to
that of a control zone or control zones, referred to herein as
Relative Intensity or RI. Results may also be expressed as the
number of copies of analyte present in the measurement volume. The
mapping of the amount of analyte detected onto other measurement
scales is a preferable embodiment for reporting results of the
inventive assay.
[0177] In addition to reporting the assay results on a continuous
scale, either directly as the amount of analyte detected or
indirectly as a measurement scale onto which the amount of analyte
detected has been mapped, the inventive assays may be used in a
"cut-off" style assay. If the detectable marker is detected in an
analyte binding zone, the amount of detectable marker detected may
be compared against a cut-off value. A cut-off value is the value
above which the test may be considered positive; that is, the
analyte of interest is present in the fluid sample to some degree
of statistical confidence. Below the cut-off value, the test is
generally considered not positive--either the analyte of interest
is not present or the inventive lateral flow assay did not detect
its presence. While a cut-off may established based upon a directly
measured value, such as the amount of analyte detected, the results
may be more meaningful if reported on an indirect, or relative,
scale.
[0178] A cut-off lateral flow assay is more desirable as the
measurement separation between a negative value and a positive
value increases. A negative value is the reported value on the
continuous scale in the case where the analyte of interest is
statistically not present. Conversely, a positive value is the
reported value on the continuous scale in the case where the
analyte of interest is statistically present. As these values
converge, the likelihood reduces of being able to statistically
tell positives and negatives apart.
[0179] Also desirable is a cut-off lateral flow with increased
precision at the cut-off. When there is less variation at the
chosen cut-off, it is more likely that a positive can be accurately
considered a positive and a negative be accurately considered a
negative.
[0180] Assay results may be mapped onto either a "relative,"
discussed above, or an "absolute" scale. Absolute scales are
measured in actual physical units, such as number of copies of
analyte per milliliter of fluid. Measurement in the absolute scale
may be preferable in testing for certain diseases or conditions,
such as tests for cancer markers, such as for PSA or hormones such
as TSH. In such preferable embodiments, the result may be expressed
in units, such as ng/ml. Accordingly, the control zones may have
value assigned concentrations of control agent. In an extension of
the relative measurement concept, the density of reflectance (DR)
values of a series of standards of known analyte concentration may
be measured and the intensities relative to the controls (RI
values) calculated as previously described. The RI values may then
be plotted against analyte concentration to construct a standard
curve in which the RI values are assigned concentration values of
the analyte of interest. The RI of a sample may then be read on
this value assigned standard curve, yielding a result labeled in
the desired units.
[0181] Many circumstances may affect the absolute reactivity of
lateral flow assays, including, but not limited to, reagent flow
variations, manufacturing-derived variations, operator induced
variations, environmentally induced variations and sample effects.
With conventional lateral flow assays, any of these variations may
act to repress or arguably enhance reactivity of one strip over
another, resulting in possible false negative or false positive
results. Not controlling for these or other variations may result
in significant imprecision, non-reproducibility, lack of
sensitivity and lack of specificity of the tests.
[0182] Lateral flow assays are also subject to a number of
interferences which might affect the absolute amount of binding of
either analyte binding agent or control agent to the test zones.
Influencing factors may include: 1) variability in the release of
the first analyte binding agent or the control agent from a
conjugate pad, 2) device to device variation in the non-specific
binding of the analyte binding population to the test strip, 3)
variability in the movement of the analyte binding population
through or along the test strip during the assay due to variations
in reagent flow rates between when a portion of the strip is dry
and when a portion of the strip is wet, variations in the pore size
of the test strip or membrane strip materials or non-specific
aggregation of the analyte binding agent. Variability of absolute
measurements of binding due to these or other factors may therefore
be unacceptably high in conventional lateral flow assays.
[0183] The use of control zones on test strips is also described in
greater detail in application Ser. No. 09/198,118, filed Nov. 23,
1998 and application Ser. No. 09/638,668, filed Aug. 14, 2000,
which are each incorporated herein by reference.
EXAMPLES
[0184] 1. Construction of Test Strip
[0185] In this example, the construction of a test strip having a
design as illustrated in FIGS. 1 and 7 is described. Backed sheets
of nitrocellulose, for example, Millipore STHF or HF 90
nitrocellulose (4.8 cm.times.20 cm) were coated by longitudinally
dispensing one antigen test band and two control bands onto the
nitrocellulose using a Biodot Quanti-3000 XYZ Dispensing Platform
with Biojets operating at a frequency of 180 Hz., 20.83 nl/drop and
0.75 .mu.l/cm. The nitrocellulose sheets were then dried for one
hour at 37.degree. C. in a forced air incubator. Coated
nitrocellulose sheets were stored desiccated at room temperature in
foil pouches.
[0186] Gelman 8980 glass fiber pads, for use as conjugate pads,
were preblocked by dipping in a solution of PBS containing 10 mg/ml
BSA, 1% (w/v) Triton X-100, 2.5% (w/v) sucrose, 0.3% (w/v)
polyvinyl pyrrolidone K-30 and 2 mg/ml rabbit IgG. The preblocked
pads were then dried for two hours in a forced air incubator. A
solution of control and test conjugates in PBS containing 10 mg/ml
BSA, 1% (w/v) Triton X-100, 2.5% (w/v) sucrose, 0.3% (w/v)
polyvinyl pyrrolidone K-30 and 2 mg/ml rabbit IgG was
longitudinally dispensed on the preblocked conjugate pads using a
Biodot Quanti-3000 XYZ Dispensing Platform with Biojets operating
at a frequency of 120 Hz., 104.17 nl/drop and 2.5 .mu.l/cm. The
conjugate pads were coated with conjugate in patterns of from one
to four lines per cm with one pattern coated on each 1.3
cm.times.20 cm pad. Coated conjugate pads were vacuum dried at 2
Torr for two hours at room temperature.
[0187] Cytosep 1662 sheets, for use in preparing sample pads, were
preblocked by dipping in a solution of PBS 10 mg/ml BSA, 1% (w/v)
Triton X-100, 2.5% (w/v) sucrose and 0.3% polyvinyl pyrrolidone
K-30. The sheets were then dried for two hours in a forced air
incubator. After drying sheets were slit to strips 7.5 mm wide
using a G&L Precision Die Cutting Drum Slitter.
[0188] Cytosep 1662 sheets, for use in preparing conjugate buffer
pads, were preblocked by dipping in a solution of PBS 10 mg/ml BSA,
1% (w/v) Triton X-100, 2.5% (w/v) sucrose, 0.3% polyvinyl
pyrrolidone K-30, 2 mg/ml Rabbit IgG, 1 mg/ml Goat IgG and 0.33
mg/ml heterophyllic blocking reagent 1 (HBR-1) then drying for two
hours in a Forced air incubator. The sheets were then slit to
strips 0.75 cm wide using a G&L Precision Die Cutting Drum
Slitter and further cut to 0.75 cm.times.1.2 cm pads using a Biodot
Guillotine cutter.
[0189] Test strips were prepared by affixing one 4.8 cm.times.20 cm
backed nitrocellulose sheet, and one 1.3 cm.times.20 cm coated
preblocked conjugate pad onto one adhesive coated 0.010" thick 6
cm.times.20 cm vinyl backing sheet (G&L Precision Die Cutting).
One 0.75 cm.times.20 cm sample pad was then affixed to the
nitrocellulose using double sided adhesive. Strips 0.5 cm wide were
cut from the assembled sheet with a Kinematics Automation Matrix
2360 Guillotine Cutter. To assemble the test strip into a test
cartridge, illustrated in FIGS. 5A and 5B, the strip was placed in
the bottom half of the holder and a 0.6 cm.times.2.7 cm absorbent
pad was placed over the top of the strip. A 0.75 cm.times.1.2 cm
preblocked conjugate buffer pad was then placed over the conjugate
pad and aligned with the bottom of the strip and the pins of the
top half of the holder aligned with the holes of the bottom half
and the holder tightly pressed together.
[0190] 2. Thyroid Stimulating Hormone (TSH) Assay
[0191] Strips used in this example were coated with 3 mg/ml rabbit
anti-dinitrophenyl (anti-DNP) in the high control band and 0.8
mg/ml rabbit anti-dinitrophenyl in the low control band and 4 mg/ml
affinity goat anti-TSH in the antigen band on Millipore SRHF
nitrocellulose. The order of the bands on the strip was low control
zone closest to the sample addition pad, high control zone farthest
from the sample addition pad (closest to the buffer addition pad)
and antigen band (anti-TSH) between the low control zone and the
high control zone. Nitrocellulose sheets were coated and strips
prepared as in Example 1.
[0192] Preblocked conjugate pads were coated with a mixture of 0.2
volumes of Anfi-DNP-32 nm gold conjugate (OD 520 nm approximately
83) and 0.13 volumes of monoclonal anti-TSH 32-nm gold conjugate
(OD 520 nm approximately 102) in a total of four volumes of PBS
containing 10 mg/ml BSA, 1% (w/v) Triton X-100, 2.5% sucrose and
0.3% (w/v) polyvinyl pyrrolidone K-30. The mixture was dispensed
onto preblocked conjugate pads as described in Example 1.
[0193] The assay was carried out by placing the cassette on the lab
bench and then adding 40 .mu.l of release buffer (5.5.times.PBS, 10
mg/ml BSA, 0.025% casein, 0.325% Tween 20, 2 mM EDTA, 0.1% sodium
azide) containing 160 pg/ml BSA-DNP to the sample addition port of
the cassette. The cassette was immediately placed in a ReLIA.TM.
machine set up to run and read the ReLIA.TM. assay for the
detection of TSH. At the prompts, sample number and assay time were
entered, triggering the sample addition clock. After a time
sufficient for prewetting of the strip to a point distal to the low
control zone (56 seconds, machine time constant of 40), the machine
prompted the user to add 150 .mu.l of the samples shown in FIG. 8
to the conjugate buffer port of the cassette. Strip temperature was
set to 30.degree. C. and the strips were read after 20 minutes.
Relative intensity values of the samples were generated by
calculating the ratio of the density of reflectance of the antigen
band to the density of reflectance of the high control band.
[0194] Table 1 below provides a definition for the various figure
headings describing the test results shown in FIG. 8 as well as in
FIG. 10.
1TABLE 1 HC(Dr) Raw density of reflectance value for the high
control band LC (Dr) Raw density of reflectance value for the low
control band HC/LC Ratio of the high control band to the low
control band. (this ratio is used as a quality control check for
each individual strip run) Specimen/ Ratio is the actual value used
by the software to HC generate the result. Using this RI ratio
helps to normalize strip to strip variability. .mu.IU/mL Micro
international units per milliliter - the quantitative level of PSH
in the sample calculated from the assay standard curve
[0195] As shown in FIG. 9, a standard curve was calculated from the
data summarized therein relating the RI value given by a TSH
standard to the TSH concentration of the standard. This standard
curve was then used to determine the mean TSH concentration of an
unknown sample, the standard deviation on the mean and percent CV,
using sixteen strips, each from a different coated nitrocellulose
sheet, from a single lot of the ReLIA.TM. TSH assay. As shown in
FIG. 9, the percent coefficient of variation on the mean TSH
concentration of 8.87 micro International units per milliliter,
determined by the ReLIA.TM. TSH assay, was 5.2% demonstrating the
high reproducibility of the RELIA.TM. TSH assay.
[0196] 3. Prostate Specific Antigen (PSA) Assay
[0197] Strips used in this example were coated with 3 mg/ml rabbit
anti-dinitrophenyl (anti-DNP) in the high control band and 1.0
mg/ml rabbit anti-dinitrophenyl in the low control band and 4 mg/ml
affinity goat anti-PSA in the antigen band on Millipore HF 135
nitrocellulose. The order of the bands on the strip was low control
zone closest to the sample addition pad, high control zone farthest
from the sample addition pad (closest to the buffer addition pad)
and antigen band (anti-PSA) between the low control zone and the
high control zone. Nitrocellulose sheets were coated and strips
prepared as in Example 1.
[0198] Preblocked conjugate pads were coated with a mixture of 0.2
volumes of Anti-DNP-32 nm gold conjugate (OD 520 nm approximately
83) and 0.14 volumes of monoclonal anti-PSA 32-nm gold conjugate
(OD 520 nm approximately 106) in a total of four volumes of PBS
containing 10 mg/ml BSA, 1% (w/v) Triton X-100, 2.5% sucrose and
0.3% (w/v) polyvinyl pyrrolidone K-30. The mixture was dispensed
onto preblocked conjugate pads as described in Example 1.
[0199] The assay was carried out by placing the cassette on the lab
bench and then adding 40 .mu.l of release buffer (5.5.times.PBS, 10
mg/ml BSA, 0.025% casein, 0.325% Tween 20, 2 mM EDTA, 0.1% sodium
azide) containing 160 .mu.g/ml BSA-DNP to the sample addition port
of the cassette. The cassette was immediately placed in a ReLIA.TM.
machine set up to run and read the ReLIA.TM. assay for the
detection of PSA. At the prompts, sample number and assay time were
entered, triggering the sample addition clock. After a time
sufficient for prewetting of the strip to a point distal to the low
control zone (56 seconds, machine time constant of 40), the machine
prompted the user to add 150 .mu.l of the samples shown in FIG. 10
to the conjugate buffer port of the cassette. Strip temperature was
set to 30.degree. C. and the strips were read after 15 minutes.
Relative intensity values of the samples were generated by
calculating the ratio of the density of reflectance of the antigen
band to the density of reflectance of the high control band.
[0200] As shown in FIG. 11, a standard curve was calculated from
the data summarized therein relating the RI value given by a PSA
standard to the PSA concentration of the standard. This standard
curve was then used to determine the mean PSA concentration of an
unknown sample, the standard deviation on the mean and the percent
CV, using sixteen strips, each from a different coated
nitrocellulose sheet, from a single lot of the ReLIA.TM. PSA assay.
As shown in FIG. 11, the percent coefficient of variation on the
mean PSA concentration of 9.00 nanograms per milliliter, determined
by the ReLIA.TM. PSA assay, was 8.9% demonstrating the high
reproducibility of the ReLIA.TM. PSA assay.
[0201] 4. ReLIA TSH (Thyroid Stimulating Hormone) Stop Flow Assay,
Sample added at Top and Bottom
[0202] In this example, the construction of the test strip used was
as follows. In general, the test strip used has a design as
illustrated in FIGS. 1 and 7. Backed sheets of nitrocellulose, for
example, Millipore STHF or HF 90 nitrocellulose ( 4.8 cm.times.20
cm) were coated by longitudinally dispensing one antigen test band
and two control bands onto the nitrocellulose using a Biodot
Quanti-3000 XYZ Dispensing Platform with Biojets operating at a
frequency of 180 Hz., 20.83 nl/drop and 0.75 .mu.l/cm. The
nitrocellulose sheets were then dried overnight at 37.degree. C. in
a forced air incubator. Coated nitrocellulose sheets were stored
desiccated at room temperature in foil pouches.
[0203] Gelman 8980 glass fiber pads, for use as conjugate pads,
were preblocked by dipping in a solution of 10 mM Sodium Borate pH
9.0 containing 0.1% polyethylene glycol (MW 20000) and 5%
Trehalose. The preblocked pads were then dried for two hours in a
forced air incubator. A solution of control and test conjugates, in
10 mM Sodium Borate pH 9.0 containing 0.1% polyethylene glycol (MW
20000) and 5% Trehalose, was longitudinally dispensed on the
preblocked conjugate pads using a Biodot Quanti-3000 XYZ Dispensing
Platform with Biojets operating at a frequency of 120 Hz., 104.17
nl/drop and 2.5 .mu.l/cm. The conjugate pads were coated with
conjugate in patterns of from one to four lines per cm with one
pattern coated on each 1.3 cm.times.20 cm pad. Coated conjugate
pads were vacuum dried at 2 Torr for two and one half hours at room
temperature.
[0204] Cytosep 1662 sheets, for use in preparing sample pads, were
preblocked by dipping in a solution of PBS 10 mg/ml BSA, 1% (w/v)
Triton X-100, 2.5% (w/v) sucrose and 0.3% polyvinyl pyrrolidone
K-30. The sheets were then dried for two hours in a forced air
incubator. After drying sheets were slit to strips 7.5 mm wide
using a G&L Precision Die Cutting Drum Slitter.
[0205] Cytosep 1662 sheets, for use in preparing conjugate buffer
pads, were preblocked by dipping in a solution of PBS 10 mg/ml BSA,
1% (w/v) Triton X-100, 2.5% (w/v) sucrose, 0.3% polyvinyl
pyrrolidone K-30, 2 mg/ml Rabbit IgG, 1 mg/ml Goat IgG and 0.33
mg/ml heterophyllic blocking reagent 1 (HBR-1) then drying for two
hours in a Forced air incubator. The sheets were then slit to
strips 0.5 cm wide using a G&L Precision Die Cutting Drum
Slitter and further cut to 0.5 cm.times.1.2 cm pads using a Biodot
Guillotine cutter.
[0206] Test strips were prepared by affixing one 4.8 cm.times.20 cm
backed nitrocellulose sheet, and one 1.3 cm.times.20 cm coated
preblocked conjugate pad onto one adhesive coated 0.010" thick 6
cm.times.20 cm vinyl backing sheet (G&L Precision Die Cutting).
One 0.75 cm.times.20 cm sample pad was then affixed to the
nitrocellulose using double sided adhesive. Strips 0.5 cm wide were
cut from the assembled sheet with a Kinematics Automation Matrix
2360 Guillotine Cutter. To assemble the test strip into a test
cartridge, illustrated in FIGS. 5A and 5B, the strip was placed in
the bottom half of the holder and a 0.6 cm.times.2.7 cm absorbent
pad was placed over the top of the strip. A 0.5 cm.times.1.2 cm
preblocked conjugate buffer pad was then placed over the conjugate
pad and aligned with the bottom of the strip and the pins of the
top half of the holder aligned with the holes of the bottom half
and the holder tightly pressed together.
[0207] Strips used in this example were coated with 500 .mu.g/ml
Dinitrophenyl Bovine Serum Albumin (BSA-DNP) in the high control
band, 100 .mu.g/ml BSA-DNP in the low control band and a 4 mg/ml
Affinity Goat anti-TSH in the antigen band. The order of the bands
on the strip was low control zone closest to the conjugate pad,
antigen band between the low control zone and the high control zone
and the high control zone farthest from the conjugate pad and
closest to the absorbent pad. Nitrocellulose sheets were coated and
strips prepared as described above.
[0208] Conjugate pads preblocked with 2 mM Sodium Borate pH 9.0
containing 5% Trehalose were coated with a mixture of Anti-DNP
conjugate [Rabbit anti-DNP (2.times.)] -30 nm gold and anti-TSH
conjugate [Monoclonal anti-TSH (2.times.)]-30 nm gold. This was
accomplished by mixing 0.3 volumes of the anti-DNP stock conjugate
solution (OD 520 approximately 100) and 0.7 volumes of the anti-TSH
conjugate (OD 520 approximately 124) with two volumes 2 mM Sodium
Borate pH 9.0 containing 5% Trehalose and one volume 2 mM Sodium
Borate pH 9.0. The mixture was dispensed onto preblocked conjugate
pads as described above.
[0209] The assay was carried out by placing the cassette on the lab
bench and then adding 50 .mu.l of the sample to the sample pad
through the proximal port of the sample cassette. The cassette
containing the strip was placed in a ReLIA.TM. machine set up to
run and read the ReLIA.TM. assay for the detection of TSH. At the
prompt additional 100 .mu.l of the sample was added to the distal
sample port of the cassette. Assay temperature was set at
30.degree. C. and the strips were read after 20 minutes. Relative
intensity (RI) values of the samples were calculated by dividing
the density of reflectance of the sample (test) band by the density
of reflectance of the high control band. A standard curve for the
TSH assay was calculated from RI values of TSH standards and
programmed into the ReLIA.TM. machine using a 4 parameter logistic
curve fit.
[0210] As shown in FIG. 12, the performance of the ReLIA.TM. TSH
assay when sample was added to both the top and bottom ports was
compared to that when sample was added only to the bottom port. The
data demonstrate that the precision on the high control density of
reflectance was higher when sample was added to the top and bottom
ports versus the bottom port alone. This translated into precision
on the measured level of TSH which was at least equivalent to that
obtained when sample was added to the bottom port alone and, in
most cases, better.
[0211] In FIG. 13 the performance of the ReLIA.TM. TSH assay using
quality control samples representing normal (approximately 1
.mu.IU/mL), borderline elevated (approximately 5 .mu.IU/mL) and
elevated (approximately 25 .mu.IU/mL) levels of TSH is displayed.
The assay was highly reproducible at all three TSH levels with CV
values under 9%.
[0212] As shown in FIG. 14, when the assay protocol employing
sample addition from both the upper and lower ports was used,
ReLIA.TM. values for TSH in patient samples, calculated by the
machine from the standard curve, correlated well with TSH levels
measured by the reference method.
[0213] It will be apparent to those skilled in the art that various
modifications and variations can be made in the apparatus and
methods of the present invention without departing from the spirit
or scope of the invention. Thus, it is intended that the present
invention cover the modifications and variations of this invention
provided they come within the scope of the appended claims and
their equivalents. Additionally, the following examples are
appended for the purpose of illustrating the claimed invention, and
should not be construed so as to limit the scope of the claimed
invention.
* * * * *